For immune diseases including allergy, cancer, and autoimmune diseases there is a pivotal role of regulation for the immune balance regulated by Th1/Th2/Th17/Treg cells and its application to development of novel immune therapies.
Th1 cells, (Type 1 helper T cells) are characterized by the production of proinflammatory cytokines like IFN-γ, IL-2, and TNF-β. Th1 cells are involved in cell-mediated immunity. The cytokines produced by Th1 cells stimulate the phagocytosis and destruction of microbial pathogens. Several chronic inflammatory diseases have been described as Th1 dominant diseases i.e. multiple sclerosis, diabetes, and rheumatoid arthritis.
Th2 cells (Type 2 helper T cells) are characterized by the production of IL-4, IL-5, IL-9, IL-10, and IL-13. Th2 cells are thought to play a role in allergy responses. Cytokines like IL-4 generally stimulate the production of antibodies. IL-5 stimulates eosinophil responses, also part of the immune response. Atopy and allergy are thought to be Th2 dominant conditions.
The imbalance of Th1/Th2 or Th17/Treg immunity becomes the cause of various immune diseases.
Allergy is considered to be a hypersensitive reaction to proteins in the environment. Allergens are antigens to which atopic patients respond with IgE antibody responses subsequently leading to allergic reactions. Antigens in the complexes or fusion proteins can be environmental allergens (e.g. house dust mite, birch pollen, grass pollen, cat antigens, cockroach antigens), or food allergens (e.g. cow milk, peanut, shrimp, soya), or a combination of both. IgE molecules are important because of their role in effector cell (mast cell, basophiles and eosinophiles) activation. It is generally accepted that IgE also plays an important role in the induction phase of allergic diseases, by up-regulating the antigen capture potential of B cells and dendritic cells (DC), both through low affinity (CD23) and high affinity receptors (FcεRI). The negative functions of IgE antibodies can be counteracted by allergen specific IgG antibodies. e.g. because they direct the immune response away from B cells to monocytes and DC and they are able to down regulate IgE receptor mediated activation of effector cells through co-cross linking the FcεRI with FcγRIIb (CD32b) on these cells. In addition, they compete with IgE molecules for allergen binding sites. Allergies therefore can be treated, cured and prevented by the induction of allergen specific IgG molecules especially IgG1.
IgG molecules have a serum half-life of approximately 3 weeks as compared to roughly 3 days for IgE molecules. IgE molecules are induced by the interaction between (naïve) B cells and Th2 cells which provide the IL-4 and IL-13 together with CD40L expression necessary to induce a class switch to IgE in memory B cells and plasma cells. In contrast, Th1 cells, which produce IFN-γ and IL-2, induce a class switch to IgG. Therefore, induction of Th1, rather than Th2 helper T cell responses against allergens, is beneficial for the prevention, treatment and cure of allergic diseases.
To date several forms of active vaccination using allergens are used. The most common is the so called “immunotherapy”, which depends on frequent immunizations with relatively high concentrations of allergens. This technique is only moderately effective in a minority of allergic diseases such as bee venom allergy and in some cases of rhinitis and conjunctivitis, and recently some reports have shown effectiveness in milder forms of asthma and atopic dermatitis. More recently rush immunotherapy, where increasing amounts of allergen are injected in a rather short time frame, has been applied with slightly better results. Usually the subcutaneous route is used for administration of the allergens, but recently this route has been compared to oral application or even local application, the results are generally positive but not always consistent. A different technique for immunotherapy is the one described by Saint-Remy (EP0178085 and EP0287361), which makes use of autologous IgG antibodies which are in vitro complexed to the relevant allergens. This technique allows far smaller amounts of allergen to be applied with fewer side effects.
The mechanism behind these therapies is unclear. In the classical therapy there seems to be a beneficial effect if the therapy induces an increase in specific IgG antibodies, although not every significant increase of specific IgG is correlated with successful immunotherapy. A possible argument why this is the case is the relatively low affinity of IgG antibodies for CD32 on B cells, monocytes and mast cells. The Saint-Remy approach selects the specific IgG antibodies from the patient, which are subsequently mixed with relevant allergens in vitro. This way they assure that the allergen cannot react freely with cells or other antibody isotypes on cells such as IgE on mast cells. In addition they claim that anti-idiotypic antibodies are raised against the specific IgG molecules, which in the future will prevent allergy.
In WO 97/07218 Allergen-anti-CD32 Fusion Proteins are described. In this publication the problems with isolating specific IgG molecules and the low affinity of these IgG antibodies for CD32 are circumvented and the risk factors of classical immunotherapy, which uses complete “IgE binding” allergens, are reduced. However, the claimed induction of Th1 memory responses due to solely directing the anti-CD32 containing vaccine to dendritic cells is not substantiated.
WO2007098934A1 describes molecules capable of binding to TLR9 and to CD32 comprising at least one epitope of at least one antigen, its production and its use as medicament, especially for the treatment of allergies.
Immunoregulation may work not only in the case of allergic diseases, but also in a series of other diseases.
Enhancing the immune response to infectious agents, such as microbial pathogens, is the goal of prophylactic or therapeutic anti-infectious immunotherapy.
In tumor immunotherapies there is also the goal to use tumor antigen specific T helper type 1 (Th1) cells in addition to cytotoxic T lymphocytes (CTL).
Cancer known medically as a malignant neoplasm, is a broad group of various diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the body through the lymphatic system or bloodstream. Not all tumors are cancerous. Benign tumors do not grow uncontrollably, do not invade neighboring tissues, and do not spread throughout the body. There are over 200 different known cancers that afflict humans.
Determining what causes cancer is complex. Many things are known to increase the risk of cancer, including tobacco use, certain infections, radiation, lack of physical activity, obesity, and environmental pollutants. These can directly damage genes or combine with existing genetic faults within cells to cause the disease. Approximately five to ten percent of cancers are entirely hereditary.
Cancer can be detected in a number of ways, including the presence of certain signs and symptoms, screening tests, or medical imaging. Once a possible cancer is detected it is diagnosed by microscopic examination of a tissue sample. Cancer is usually treated with chemotherapy, radiation therapy and surgery. The chances of surviving the disease vary greatly by the type and location of the cancer and the extent of disease at the start of treatment. While cancer can affect people of all ages, and a few types of cancer are more common in children, the risk of developing cancer generally increases with age. In 2007, cancer caused about 13% of all human deaths worldwide (7.9 million). Rates are rising as more people live to an old age and as mass lifestyle changes occur in the developing world.
Since the immune system responds to the environmental factors it encounters on the basis of discrimination between self and non-self, many kinds of tumor cells that arise as a result of the onset of cancer are more or less tolerated by the patient's own immune system since the tumor cells are essentially the patient's own cells that are growing, dividing and spreading without proper regulatory control.
Immune tolerance or immunological tolerance is the process by which the immune system does not attack an antigen. In natural or self-tolerance, the body does not mount an immune response to self-antigens. It occurs in three forms: central tolerance, peripheral tolerance and acquired tolerance
Central Tolerance1:
Central tolerance occurs during lymphocyte development and operates in the thymus and bone marrow. Here, T and B lymphocytes that recognize self-antigens are deleted before they develop into fully immunocompetent cells, preventing autoimmunity. This process is most active in fetal life, but continues throughout life as immature lymphocytes are generated.
Peripheral Tolerance2:
Peripheral tolerance is immunological tolerance developed after T and B cells mature and enter the periphery. The T cells that leave the thymus are relatively but not completely safe. Some will have receptors (TCRs) that can respond to self-antigens that are present in such high concentration that they can bind to “weak” receptors the T cell did not encounter in the thymus (such as, tissue-specific molecules like those in the islets of Langerhans, brain or spinal cord). Those self-reactive T cells that escape intrathymic negative selection in the thymus can inflict cell injury unless they are deleted or effectively muzzled in the peripheral tissue. Several feedback mechanism to silence such potentially auto reactive T cells are known to exist. They include following: Anergy, Activation-induced cell death, Peripheral suppression
Acquired or Induced Tolerance3:
Acquired or induced tolerance refers to the immune system's adaptation to external antigens characterized by a specific non-reactivity of the lymphoid tissues to a given antigen that in other circumstances would likely induce cell-mediated or humoral immunity. One of the most important natural kinds of acquired tolerance is immune tolerance in pregnancy, where the fetus and the placenta must be tolerated by the maternal immune system.
Immunotherapy Targeting Tumor Associated Antigens:
Cancer immunotherapy is the use of the immune system to reject cancer. The main premise is stimulating the patient's immune system to attack the malignant tumor cells that are responsible for the disease. This can be done either through active immunization of the patient (e.g., by administering a cellular cancer vaccine, such as Provenge, Dendreon, Seattle, Wash., US)4, in which case the patient's own immune system is trained to recognize tumor cells as targets to be destroyed, or through the administration of therapeutic antibodies as drugs, in which case the patient's immune system is recruited to destroy tumor cells by the therapeutic antibodies. Another approach for activating the patient's immune system against tumors is to make use of so called tumor associated antigens (TAA's), which are self-proteins which are to some extend expressed on healthy normal cells, but overexpressed on tumor cells or they comprise of cell hormones/growth factors to which the tumor cells proliferate5. These TAAs are formulated and presented to the body in an immunogenic fashion such that the immune system will build a response despite the fact that these proteins are self. Obviously this approach will only be useful for TAAs against which the patient has developed peripheral or acquired tolerance. When the T and B cells recognizing the TAA have been deleted from the immunological repertoire, active cancer immunotherapy is not an option.
Gastrin:
An example of an autoantigen (hormone/growth factor) that may be used as target for treatment of gastro intestinal cancers such as pancreatic cancer is little gastrin (G17)6-9. In addition, neutralization of G17 may also be beneficial in any gastrin related disease condition, including gastric ulcers, Gastro Esophageal Reflux Disease (GERD)10, since the pH of the stomach is regulated by gastrin, and for End Stage Renal Failure (ESRF)11, since gastrin circulates at higher than normal concentrations in ESRF patients.
U.S. Pat. No. 5,023,077 describes immunogenic compositions and methods for the treatment and prevention of gastric and duodenal ulcer disease, which immunogenic compositions are based on gastrin peptides, which are coupled to an immunogenic carrier, such as diptheria toxoid, tetanus toxoid, keyhole limpet hemocyanin or bovine serum albumin.
Gastrin has several important functions in the gastrointestinal tract, the two most important being stimulation of acid secretion and stimulation of the growth of cells in the gastrointestinal tract. The hormone exists in at least two molecular forms, heptadecagastrin, the so-called little gastrin (“G17”), and tetratriacontagastrin (“G34”) named according to the number of amino acid residues (“AA's”) in each molecule, wherein the G17 constitutes the 17 amino terminal (“N-terminal”) residues of G34.
U.S. Pat. No. 5,609,870 describes the preparation of an anti-G17 immunogen which raises antibodies in a mammal against its own G17 which do not react with G34 comprising conjugating a peptide which consists of a sequence corresponding to a fragment of the N-terminal amino acid sequence of G17 up to amino acid residue number 12 by its C-terminus to a spacer peptide which is conjugated to an immunogenic carrier, such as diphtheria toxoid, tetanus toxoid, keyhole limpet hemocyanin, and bovine serum albumin.
Immune Balance:
The immune balance regulated by Th1/Th2/Th17/Treg cells plays a significant part in the development of immune therapies.
In autoimmune diseases there is a need to regulate the Th1/Th2/Th17/Treg imbalance, i.e. in conditions where the immune system attacks self-tissue.
The Role of TLR9:
Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system. They are single, membrane-spanning, non-catalytic receptors usually expressed on the cell surface and in the endocytic compartment of sentinel cells such as macrophages and dendritic cells. TLR's recognize pathogen-associated molecular patterns (PAMPs), structurally conserved molecules, derived from microbes and initiate signalling to induce production of cytokines necessary for the innate immunity and subsequent adaptive immunity.
The various TLRs exhibit different patterns of expression. This gene is preferentially expressed in immune cell rich tissues, such as spleen, lymph node, bone marrow and peripheral blood leukocytes.
Thirteen TLRs (named simply TLR1 to TLR13) have been identified in humans and mice together, and equivalent forms of many of these have been found in other mammalian species. However, not every TLR receptor in mice is also found in humans or vice versa. In addition, not for every TLR receptor the ligand and function is known, e.g. TLR10 is orphan receptor with unknown function.
Activation of TLR receptors has been used for the treatment of various diseases e.g activation of TLR9 by pharmaceutical products has been shown to be beneficial in treatment of allergy and oncology. Studies in mice and human indicate that the natural ligands of TLR9 are unmethylated CpG sequences in DNA molecules. CpG sites are relatively rare (˜1%) on vertebrate genomes in comparison to bacterial genomes or viral DNA. TLR9 is expressed by numerous cells of the immune system such as dendritic cells, B lymphocytes, monocytes and natural killer (NK) cells. However in healthy humans the TLR9 is expression is restricted to plasmacytoid dendritic cells (pDCs) and B cells. The expression is intracellularly, within the endosomal compartments and functions to alert the immune system of viral and bacterial infections by binding to DNA rich in CpG motifs. However under pathological conditions TLR9 expression has been reported on the cell surface of cells as well12-14.
Many different synthetic TLR9 agonist molecules have been reported. The agonistic ligands (TLR9 activating) have been classified into three groups:
The group consisting of CpG class A, in particular CpG-A (D)15 oligodeoxynucleotides (ODN), also known as “D”-type ODN. Such TLR9 agonists induce a strong IFNa induction and minimal maturation of dendritic cells, and are herein called “group 1” TLR9 ligand. An example is ODN221616:
(SEQ ID 48)GGGGGACGATCGTCGGGGGG
The group consisting of CpG class B, in particular CpG-B (K)17 oligodeoxynucleotides (ODN), also known as “K”-type ODN. Such TLR9 agonists induce a weak IFNa induction and maturation of dendritic cells, and are herein called “group 2” TLR9 ligand. An example is ODN200618;19:
(SEQ ID 49)TCGTCGTTTTGTCGTTTTGTCGTT
The group consisting of CpG class C, also known as CpG-C20 oligodeoxynucleotides (ODN). Such TLR9 agonists induce IFNa and maturation of immature dendritic cells, and are herein called “group 3” TLR9 ligand. An example is ODNM36221:
(SEQ ID 69)TCGTCGTCGTTCGAACGACGTTGAT
All of the ligands for TLR9 described to date are based on nucleotides. Although antibodies specific for TLR9 have been reported and used to demonstrate the presence and location of the receptor, these molecules have not been described as ligands for TLR9, there was no report of any TLR9 activating or inhibiting activity.
The Role of CD32:
CD32 is strongly expressed on monocytes/dendritic cells and B cells and thus such molecules are designed to direct the immune response to these important immunological cells, with the intention to prevent antigen presentation by the B cells, while promoting antigen presentation by especially dendritic cells (DCs), the latter leads to induction of Th1 responses against the antigen, when sufficiently stimulated. There are at least two types of DCs: myeloid (mDC) and plasmacytoid dendritic cells (pDC), which has led to the new concept of DC1 and DC2 cells. In this concept DC1 cells promote the induction of Th1 cell development after antigen specific stimulation and DC2 cells support the development of Th2 cells. Monocyte derived DC (or mDC) are generally considered to be of DC1 type, whereas pDC are considered to be DC2 type. Both types of DC express CD32a and will induce an antigen specific T cell response; however it is not guaranteed that the outcome will be of Th1 type. In fact, in allergic donors Th2 responses are more likely. Importantly, the pDC express the TLR9 receptor, which binds CpG-ODNs (oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs). Activation of this receptor in the pDC leads to a very strong production of IFN-alpha and IL-12, which promotes Th1 induction and thus transforms the potential DC2 into DC1 cells.
Thus, such molecules can combine the activation of the TLR9 receptor in pDC with the specific stimulation and induction of antigen specific Th1 cells.
In tumor immunotherapies there is the particular goal to use tumor antigen specific T helper type 1 (Th1) cells in addition to cytotoxic T lymphocytes (CTL).
Coiled Coils:
Coiled coils are consisting of structural motifs in proteins, in which 2-7 alpha-helices are coiled together like the strands of a rope; dimers and trimers are the most common types. The coiled coil helixes have been used to stabilize Fv antibody fragments resulting in heterodimeric coiled-coil domains22.
The stability and folded structure of complex proteinaceous molecules is crucial when designing immunogens. It is thus the object of the invention to provide a vaccine with improved stability and structure to regulate the immune response to specific immunogens.
There is further a need to provide improved immunotherapies targeting gastrin and gastrin dependent disease conditions. It is thus the object of the invention to provide a vaccine with improved immunogenicity, stability and structure to regulate the immune response to specific gastrin epitopes.